Tec-Monterrey Presentation - iGEM

A synthetic biology approach
for the sugar cane industry
improvement:
Introducing enzyme surface
display as an alternative to
enzyme immobilization
iGEM
Tec-Monterrey

Brainstorm
Last year’s Biosensor
Sulfurafane Metabolic pathway
Curing Cancer

Different Approach
Feasable Project
Social and Economical Impact
Versatile

Mexican Sugar Cane Industry
13.5% National agricultural production
5.3 million tons of sugar
4.8 million annual tons

57 sugar mills near 277 cities inhabited
by 12 million people
450,000 direct jobs
Benefits 2.2 million

Actual context
Replacement of sugar by sweeteners like high
fructose corn syrup.
Industry of beverages
NAFTA 2008 (North American Free Trade Agreement)
Total cost of sugar production takes 80% of its sales.

Opportunities
2011
Past
sucrose sucrose
bagasse bagasse
11 million tons
(51.8% cellulose)
Imported
fructose
CO 2
emission

Sugar Cane Industry
Advances
Novel Process: Sugar Cane Juice- Fructose

However there is still
room left for optimization…
Enzyme
Overproduction
Enzyme
Purification
Enzyme
Immobilization

Analysis
Economical Impact
Social Impact
Poor handling of byproducts
Expensive downstream
processing, specifically
purification and immobilization
of enzyme

How can we help?
If we can provide a synthetic biology
approach, to improve the sugar cane
industry, then it will gain an added value
by manufacturing valuable products
High Fructose Syroup out of Sucrose
Biofuels substrates out of bagaze (cellulose)

Main Objective
Provide as a proof of concept, a genetic
construction in a model microorganism
(Escherichia coli), capable of displaying
functional enzymes (invertase and cellulase)
outside the cell.

Specific Objectives
1- Selection of a Capable Membrane Proteins
2- Selection of suitable Enzymes
3- Selection of appropriate strains
4- Design of a functional expression cassette
5-Evaluation of the expression of the constructs
6- Messurement of the chimeric enzymes activity

Membrane Proteins
PhoA SP + EstA Fusion
Origin- Pseudomonas aeruginosa
Excression Mechanism: Type V
PhoA SP- Alkalyne Phosphatase
Compatibility- Free C-terminus
Linker + EstA
Membrane Protein
1-Extracellular
Transport Ability
2-Signal Peptide
3-Compatibility
Lpp SP + OmpA Fusion
Origin- Escherichia coli
Compatibility- Free N- Terminus
Excression mechanism: Type II
Lpp SP- Native Lipoprotein

Extracellular
Cellulase
1-Structure
2-Characteristics
3-Active Site
SacC Invertase
Origin- Zymmomonas mobilis
Structure- Monomeric Structure
Characteristics- 20°-40° C, pH 2.5-7.5, 48 kDa
Free N-Terminus
Extracellular
Sucrase
RBS+signal peptide
phoA+Cellulase
CelD Cellulase
Origin- Clostridium thermocellum
Structure- Monomeric
Characteristics- Max 80° C, pH 5-8, 68 kDa
Free C-Termius

Protein Expression Systems
Rosetta Gami
BL21 SI
BL21 Star
XL1Blue
C43
Characterization AraBAD
BW27783
E. coli Strains

Expression Cassette Design
Arabinose
Induced
Constructs

Construct Expression

CelD Expression
Results
o Expected MW
fusion protein
(estA + celD) 102.5
kDa

Device Functionality

CelD + estA Activity
Absorbance @540.0 nm
REDUCING
SUGARS
0.5
0.4
0.3
0.2
0.1
0
DNS
Calibration Curve
y = 0.3085x - 0.0641
R² = 0.982
0 0.5 1 1.5 2
Concentration of Glucose(mM)
Rosetta Gami
Proportional
Colorimetric
Concentration

Rosetta
Gami
Whole-Cell
Cell Lysate
Fraction
Soluble
Insoluble

Glucose Concentration (uM)
400
350
300
250
200
150
100
50
0
T-test
Alpha = 0.05
Ho -> rejected
Whole-Cell Cellulase Activity
C- celD+estA
Whole-Cell Cellulase Activity was determined by IUPAC Filter Paper Assay, with E. coli
strain, Rosetta Gami, negative control and transformed
Suggesting

Glucose Concentration (u M)
Cellulase Activity of Cell lysates
400
350
300
250
200
150
100
50
0
T-test
Alpha = 0.05
Ho -> rejected
SOLUBLE FRACTION
C- celD+estA
Glucose Concentration (u M)
400
350
300
250
200
150
100
50
0
INSOLUBLE FRACTION
C- celD+estA
Cell Lysate Fractions Activity was determined by IUPAC Filter Paper Assay, with E. coli
strain, Rosetta Gami cellular lysate, negative control and transformed with celD+estA

We can conclude that …
Difference between negative control and
estA+celD -> Statiscally significant
Cellulase + estA …. ACTIVE
Activity in C- …. Background signal

Future Work
• Standarize the IUPAC Filter Paper Assay ->
more measurements
• LB media -> M9 media or others
• Different E. coli strains
• Another measurement methods e.g.
Benedict method , HPLC

Construct Expression

SacC Expression
Expected MW
fusion protein
(OmpA + SacC)
62.8 kDa
Results
Unclear evidence
vector expression
by SDS-PAGE
+ Coomassie blue

Device Functionality

Whole Cell SacC Activity
Enzyme assay
Enzymatic reaction using
sucrose as substrate
@ pH 5.0, @ 36°C 30 min
Quantification of
released fructose
Colorimetric assay based
on Tetrazole reduction
BL21 SI

BL21SI
t-Test (2 tails, α=0.05)
Rejects the null hypothesys
H 0 = The population means are the same
Tetrazole ---- Fructose

Conclusion
• More Specific Stain
• More measurements
• Different E. coli strains
• Another measurement methods e.g. HPLC

Human Practice
Genes in a Bottle
Molecular biology workshop
Ene.Pé notes
Myths and facts Biotech
MicroCongress

Augmented Biobricks
Synthetic biology
Real time 3D modeling of
your construct

Further Approach
Comparing Raw Data with analytic tests
Eukaryotic systems for heterologous
expression
Sustainable high fructose syrup process
Unit operaations

Thank you.